Tumor cells express certain antigens which are absent from, or present in small amounts on, their normal cellular counterparts. Most of these are differentiation antigens, shared by the tumor and various embryonic cells. Some of the antigens that appear with sufficient selectivity may serve as possible targets for therapeutic agents.
More than 40 different melanoma antigens have been defined with monoclonal antibodies, resulting in several major antigenic families with immunologically and biologically distinct characteristics. Among these families are: (1) the high molecular weight oncofetal proteins; (2) the gangliosides; (3) receptors for growth factors such as EGF, PDGF, TGF-alpha and TGF-beta, and nerve growth factor; (4) cation transport and binding proteins such as p97; (5) HLA class II antigens; (6) pigmentation-associated antigens; and (7) extracellular matrix proteins. Herlyn and Koproski, Ann. Rev. Immunol. 6: 283-308 (1988).
While preliminary studies with monoclonal antibody-based therapy and diagnosis directed toward various of these antigens are encouraging, work continues unabated in the hope that better agents and antigenic targets can be identified. Cutaneous malignant melanoma is increasing in prevalence at an alarming rate, particularly in the United States.
More recently a new family of antigens has been described on melanoma tumors. These antigens, now termed the xe2x80x9cmelanoma antigen,xe2x80x9d or MAGE family of antigens, were identified in a melanoma cell line which was lysed by a panel of autologous cytotoxic T lymphocytes (xe2x80x9cCTLsxe2x80x9d). Cells which did not express a MAGE-type antigen were not killed by the CTL, and by selecting these xe2x80x9cantigen-lossxe2x80x9d variants, six independent antigens were identified. Van den Eynde et al., Int. J. Cancer, 44: 634 (1989). A gene encoding one of the antigens, designated MZ2-E (xe2x80x9cExe2x80x9d), has been cloned and sequenced. Van der Bruggen et al., Science 254: 1643 (1991). The sequence was deposited in GenBank (accession #M77481), and comparison of the nucleotide sequence, designated xe2x80x9cMAGE-1,xe2x80x9d failed to reveal any significant homology with any sequence in data banks, including GenBank. Two additional nonidentical cDNAs were also found (MAGE-2 and MAGE-3) which were more closely related to each other than to MAGE-1, but the three were approximately equally expressed.
Smaller regions of the MAGE-1 gene were cloned and transfected into cells. These transfectants expressed antigen which was recognized by the anti-E CTLS. Thus, it appears that the gene does not encode a protein which further activates an antigen-encoding gene. Van der Bruggen, id. The sequence encoding the antigenic peptide was speculated to be within the region of overlap of the segments. See Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). The cDNAs of MAGE-2 and MAGE-3 were unable to transfer the expression of antigen E in transfection experiments. The presenting molecule for the E-antigen was thought to be HLA-A1.
The MAGE gene family has been shown by Van der Bruggen et al., id., to be expressed by a variety of different tumors and are not limited to melanomas, but they are not expressed by most normal cells. Thus, the MAGE antigens may have important implications for cancer immunotherapy. The sequence of the MAGE-1 gene was thought to be identical in both normal tissues and in tumors.
What is needed in the art is a more thorough understanding of the immunogenic tumor-rejecting epitopes of the MAGE antigens. Once the immunodominant epitopes are identified, along with their HLA restriction, more effective therapeutic protocols can be devised. The present invention fulfills these and other related needs.
This invention is based in part on the novel and unexpected observation that the previously reported gene encoding the human MAGE-1 protein encodes an additional fifty-eight amino acids at the C-terminal end. The complete human MAGE-1 protein and peptides thereof can be produced by recombinant or synthetic means and may or may not have the biological activity of the native MAGE-1 antigen, depending on the intended use. Accordingly, isolated and purified polynucleotides are described which code for the complete human MAGE-1 protein. The cDNA which codes for the fall length human MAGE-1 protein may be incorporated into a recombinant DNA vector; which in turn may be used to transform a suitable host; the host cell transformed with the vector including the cDNA can express full length human MAGE-1 protein, and the full length human MAGE-1 protein can be recovered.
This invention further concerns MAGE-1 immunogenic peptides from the C-terminus of the MAGE-1 protein which induce CTL activity. The immunogenic peptides of this invention may be identified using motifs as described in copending U.S. patent applications Ser. No. 07/926,666 and Ser. No. 08/027,146 for the various MHC class I alleles. Thus, small synthetic or recombinant peptides can be prepared which immunologically mimic MAGE-1 CTL inducing antigenic determinants. The CTL-inducing MAGE-1 peptides of the invention can be used therapeutically, for example, to induce, in the context of an appropriate MHC presenting molecule, an immunological response to tumors which express the corresponding MAGE determinants. In this manner the tumor cells can be killed or inhibited. The induction of CTLs can be accomplished in vivo or ex vivo. Thus, the MAGE-1 peptides described herein also can be formulated and administered as pharmaceutical compositions, especially when used to induce immunological responses in individuals predisposed to developing or already afflicted by a tumor which expresses MAGE-1 determinants.
In yet other embodiments the invention relates to methods for diagnosis, where the peptides of the invention are used to determine the presence in an individual of lymphocytes which are capable of a cytotoxic T cell response to MAGE-1 antigen. Typically the lymphocytes are peripheral blood lymphocytes and the individual of interest is suffering from a tumor associated with MAGE antigen. The diagnostic methods and compositions can be used in conjunction with therapeutic approaches to MAGE related diseases, and particularly the treatment of malignant melanoma.